Mutant E. coli Strain with Increased Succinic Acid Production

• US 20070184539A1
• Filed: 04/05/2007
• Published: 08/09/2007
• Est. Priority Date: 08/27/2004
• Status: Active Grant

• Current Assignee: Rice University
• Original Assignee: Rice University
• Inventors: San, Ka-Yiu, Bennett, George, Sanchez, Allen
• Granted Patent: US 7,790,416 B2
• Time in Patent Office: Days
• US Class Current: 435/136
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: C12P 7/46 Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid

Abstract

The invention relates to a mutant strain of bacteria, which either lacks or contains mutant genes for several key metabolic enzymes, and which produces high amounts of succinic acid under anaerobic conditions.

Claims

1-25. -25. (canceled)

26. A method of producing carboxylic acids in a bacterial cell comprising:

a) culturing a bacteria under anaerobic conditions said bacteria comprising a disruption of lactate dehydrogenase (ldh), alcohol dehydrogenase (adh), and a disruption of acetate kinase (ack), phosphotransacetylase (pta) or both ack-pta; and

b) isolating one or more carboxylic acids,

wherein glycolytic flux from oxaloacetate (OAA) is partitioned in a ratio between 10-40% citrate and between 90-60% malate.

27. The method of claim 26, wherein said glycolytic flux is partitioned in a ratio between about 30% citrate and about 70% malate.

28. The method of claim 26, wherein said bacteria produces greater than 1.5 moles of carboxylic acid per mole of glucose.

29. The method of claim 26, wherein said bacteria comprise an expression construct encoding pyruvate carboxylase (pyc).

30. The method of claim 29, wherein said pyc is from Lactobacillus lactis.

31. The method of claim 26, wherein said bacteria comprise an expression construct encoding citrate synthase (citZ).

32. The method of claim 31, wherein said citZ is from Bacillus subtilis.

33. A method of producing succinic acid in a bacterial cell comprising:

a) culturing a bacteria under anaerobic conditions said bacteria comprising a disruption of ldh, adh, and a disruption of ack, pta or both ack-pta; and

b) isolating succinic acid,

wherein glycolytic flux from OAA is partitioned in a ratio between 10-40% citrate and between 90-60% malate.

34. The method of claim 33, wherein said glycolytic flux is partitioned in a ratio between about 30% citrate and about 70% malate.

35. The method of claim 33, wherein said bacteria produces greater than 1.5 moles of succinic acid per mole of glucose.

36. The method of claim 33, wherein said bacteria comprise an expression construct encoding pyc.

37. The method of claim 36, wherein said pyc is from Lactobacillus lactis.

38. The method of claim 33, wherein said bacteria comprise an expression construct encoding citZ.

39. The method of claim 38, wherein said citZ is from Bacillus subtilis.

40. A method of producing succinic acid in a bacterial cell comprising:

a) culturing a bacteria under anaerobic conditions said bacteria comprising a disruption of ldh, adh, aceBAK operon repressor (iclR), and aerobic respiratory control regulator (arc), and a disruption of ack, pta or both ack-pta;

b) isolating succinic acid,

wherein glycolytic flux from OAA is partitioned in a ratio between 10-40% citrate and between 90-60% malate.

41. The method of claim 40, wherein said glycolytic flux is partitioned in a ratio between about 30% citrate and about 70% malate.

42. The method of claim 40, wherein said bacteria produces greater than 1.5 moles of succinic acid per mole of glucose.

43. The method of claim 40, wherein said bacteria comprise an expression construct encoding pyc.

44. The method of claim 43, wherein said pyc is from Lactobacillus lactis.

45. The method of claim 40, wherein said bacteria comprise an expression construct encoding citZ.

46. The method of claim 45, wherein said citZ is from Bacillus subtilis.